Expedient production of site specifically nucleobase-labelled or hypermodified RNA with engineered thermophilic DNA polymerases.

Innovative approaches to controlled nucleobase-modified RNA synthesis are urgently needed to support RNA biology exploration and to synthesize potential RNA therapeutics. Here we present a strategy for enzymatic construction of nucleobase-modified RNA based on primer-dependent engineered thermophilic DNA polymerases - SFM4-3 and TGK. We demonstrate introduction of one or several different base-modified nucleotides in one strand including hypermodified RNA containing all four modified nucleotides bearing four different substituents, as well as strategy for primer segment removal. We also show facile site-specific or segmented introduction of fluorophores or other functional groups at defined positions in variety of RNA molecules, including structured or long mRNA. Intriguing translation efficacy of single-site modified mRNAs underscores the necessity to study isolated modifications placed at designer positions to disentangle their biological effects and enable development of improved mRNA therapeutics. Our toolbox paves the way for more precise dissecting RNA structures and functions, as well as for construction of diverse types of base-functionalized RNA for therapeutic applications and diagnostics.

Circulating Cell-Free DNA-Based Methylation Pattern in Saliva for Early Diagnosis of Head and Neck Cancer.

Head and neck cancer (HNC) remains one of the leading causes of mortality worldwide due to tumor diagnosis at a late stage, loco-regional aggression, and distant metastases. A standardized diagnostic procedure for HNC is a tissue biopsy that cannot faithfully portray the in-depth tumor dynamics. Therefore, there is an urgent need to develop simple, accurate, and non-invasive methods for cancer detection and follow-up. A saliva-based liquid biopsy allows convenient, non-invasive, and painless collection of high volumes of this biofluid, with the possibility of repetitive sampling, all enabling real-time monitoring of the disease. No approved clinical test for HNC has yet been established. However, epigenetic changes in saliva circulating cell-free DNA (cfDNA) have the potential for a wide range of clinical applications. Therefore, the aim of this review is to present an overview of cfDNA-based methylation patterns in saliva for early detection of HNC, with particular attention to circulating tumor DNA (ctDNA). Due to advancements in isolation and detection technologies, as well as next- and third-generation sequencing, recent data suggest that salivary biomarkers may be successfully applied for early detection of HNC in the future, but large prospective clinical trials are still warranted.

Direct Stereodivergent Olefination of Carbonyl Compounds with Sulfur Ylides.

The reactivity of phosphorus and sulfur ylides toward carbonyl compounds constitutes a well-known dichotomy that is a common educational device in organic chemistry─the former gives olefins, while the latter gives epoxides. Herein, we report a stereodivergent carbonyl olefination that challenges this dichotomy, showcasing thiouronium ylides as valuable olefination reagents. With this method, aldehydes are converted to Z-alkenes with high stereoselectivity and broad substrate scope, while N-tosylimines provide a similarly proficient entry to E-alkenes. In-depth computational and experimental studies clarified the mechanistic details of this unusual reactivity.

Nucleotides bearing aminophenyl- or aminonaphthyl-3-methoxychromone solvatochromic fluorophores for the enzymatic construction of DNA probes for the detection of protein-DNA binding.

We designed and synthesized nucleosides bearing aminophenyl- or aminonaphthyl-3-methoxychromone fluorophores attached at position 5 of cytosine or thymine and converted them to nucleoside triphosphates. The fluorophores showed solvatochromic fluorescence with strong fluorescence at 433-457 nm in non-polar solvents and very weak fluorescence at 567 nm in alcohols. The nucleosides and nucleotides also showed only negligible fluorescence in alcohols or water. The triphosphates were substrates for DNA polymerase in the enzymatic synthesis of modified DNA probes that showed only very weak fluorescence in aqueous buffer but a significant light-up and blue shift were observed when they interacted with proteins (histone H3.1 or p53 for double-stranded DNA probes or single-strand binding protein for single-stranded oligonucleotide probes). Hence, nucleotides have good potential in the construction of DNA sensors for studying protein-DNA interactions. The modified dNTPs were also transported into cells using a cyclodextrin-based transporter but they were not incorporated into the genomic DNA.

Stable and easily available sulfide surrogates allow a stereoselective activation of alcohols.

Isothiouronium salts are easily accessible and stable compounds. Herein, we report their use as versatile deoxasulfenylating agents enabling a stereoselective, thiol-free protocol for synthesis of thioethers from alcohols. The method is simple, scalable and tolerates a broad range of functional groups otherwise incompatible with other methods. Late-stage modification of several pharmaceuticals provides access to multiple analogues of biologically relevant molecules. Performed experiments give insight into the reaction mechanism.

Chemoselective γ-Oxidation of ß,γ-Unsaturated Amides with TEMPO.

A chemoselective and robust protocol for the γ-oxidation of ß,γ-unsaturated amides is reported. In this method, electrophilic amide activation, in a rare application to unsaturated amides, enables a regioselective reaction with TEMPO resulting in the title products. Radical cyclisation reactions and oxidation of the synthesised products highlight the synthetic utility of the products obtained.

2-Substituted 2'-deoxyinosine 5'-triphosphates as substrates for polymerase synthesis of minor-groove-modified DNA and effects on restriction endonuclease cleavage.

Five 2-substituted 2'-deoxyinosine triphosphates (dRITP) were synthesized and tested as substrates in enzymatic synthesis of minor-groove base-modified DNA. Only 2-methyl and 2-vinyl derivatives proved to be good substrates for Therminator DNA polymerase, whilst all other dRITPs and other tested DNA polymerases did not give full length products in primer extension. The DNA containing 2-vinylhypoxanthine was then further modified through thiol-ene reactions with thiols. Cross-linking reaction between cysteine-containing minor-groove binding dodecapeptide and DNA proceeded thanks to the proximity effect between thiol and vinyl groups inside the minor groove. 2-Substituted dIRTPs and also previously prepared 2-substituted 2'-deoxyadenosine triphosphates (dRATP) were then used for enzymatic synthesis of minor-groove modified DNA to study the effect of minor-groove modifications on cleavage of DNA by type II restriction endonucleases (REs). Although the REs should recognize the sequence through H-bonds in the major groove, some minor-groove modifications also had an inhibiting effect on the cleavage.

Brightly Fluorescent 2'-Deoxyribonucleoside Triphosphates Bearing Methylated Bodipy Fluorophore for in Cellulo Incorporation to DNA, Imaging, and Flow Cytometry.

Synthesis of cytosine, uracil, and 7-deazaadenine 2'-deoxyribonucleosides and triphosphates (dNTPs) bearing hexamethylated phenyl-bodipy fluorophore attached at position 5 of pyrimidines or at position 7 of 7-deazapurine was developed. All the title labeled nucleosides and dNTPs displayed bright green fluorescence with very high quantum yields. The modified dNmBdpTPs were good substrates to diverse DNA polymerases and were used for in vitro enzymatic synthesis of labeled DNA by primer extension or PCR. In combination with cationic cyclodextrin-peptide-based dNTP transporter, the dNmBdpTPs were successfully used for staining of genomic DNA in living cells for applications in confocal microscopy and in flow cytometry. The best performing cytosine nucleotide dCmBdpTP was used to monitor mitosis in live cells.

2-Allyl- and Propargylamino-dATPs for Site-Specific Enzymatic Introduction of a Single Modification in the Minor Groove of DNA.

A series of 2-alkylamino-2'-deoxyadenosine triphosphates (dATP) was prepared and found to be substrates for the Therminator DNA polymerase, which incorporated only one modified nucleotide into the primer. Using a template encoding for two consecutive adenines, conditions were found for incorporation of either one or two modified nucleotides. In all cases, addition of a mixture of natural dNTPs led to primer extension resulting in site-specific single modification of DNA in the minor groove. The allylamino-substituted DNA was used for the thiol-ene addition, whereas the propargylamino-DNA for the CuAAC click reaction was used to label the DNA with a fluorescent dye in the minor groove. The approach was used to construct FRET probes for detection of oligonucleotides.

2-Substituted dATP Derivatives as Building Blocks for Polymerase-Catalyzed Synthesis of DNA Modified in the Minor Groove.

2'-Deoxyadenosine triphosphate (dATP) derivatives bearing diverse substituents (Cl, NH2 , CH3 , vinyl, ethynyl, and phenyl) at position 2 were prepared and tested as substrates for DNA polymerases. The 2-phenyl-dATP was not a substrate for DNA polymerases, but the dATPs bearing smaller substituents were good substrates in primer-extension experiments, producing DNA substituted in the minor groove. The vinyl-modified DNA was applied in thiol-ene addition and the ethynyl-modified DNA was applied in a CuAAC click reaction to form DNA labelled with fluorescent dyes in the minor groove.

Solvatochromic fluorene-linked nucleoside and DNA as color-changing fluorescent probes for sensing interactions.

A nucleoside bearing a solvatochromic push-pull fluorene fluorophore (dCFL ) was designed and synthesized by the Sonogashira coupling of alkyne-linked fluorene 8 with 5-iodo-2'-deoxycytidine. The fluorene building block 8 and labeled nucleoside dCFL exerted bright fluorescence with significant solvatochromic effect providing emission maxima ranging from 421 to 544 nm and high quantum yields even in highly polar solvents, including water. The solvatochromism of 8 was studied by DFT and ADC(2) calculations to show that, depending on the polarity of the solvent, emission either from the planar or the twisted conformation of the excited state can occur. The nucleoside was converted to its triphosphate variant dCFLTP which was found to be a good substrate for DNA polymerases suitable for the enzymatic synthesis of oligonucleotide or DNA probes by primer extension or PCR. The fluorene-linked DNA can be used as fluorescent probes for DNA-protein (p53) or DNA-lipid interactions, exerting significant color changes visible even to the naked eye. They also appear to be suitable for time-dependent fluorescence shift studies on DNA, yielding information on DNA hydration and dynamics.

Radical scavenging activity of Caesalpinia spinosa.

OBJECTIVES: To assess protective effects of the tara (Caesalpinia spinosa) extract against hyaluronan (HA) degradation evoked by cupric ions and ascorbate. METHODS: Uninhibited/inhibited HA degradation was assayed by a decrease in dynamic viscosity of the HA solutions, whereas as a method rotational viscometry was used. To determine radical scavenging capacity of the tara extract, the ABTS and DPPH assays were performed. RESULTS: The results of rotational viscometry showed remarkable protective effects of the tara extract against the degradation of HA. In the ABTS and DPPH assays the IC50 values of the tara extract 1.59 and 30.8 µg/mL indicated quite high radical scavenging properties. CONCLUSION: The tara extract is an efficient antioxidant as demonstrated by the methods used.